Deciphering neural coding will require deconstructing the complex and intertwined signaling mechanisms that drive cellular excitability, synaptic plasticity, and circuit dynamics in the brain. This fundamental objective has been extremely challenging because unraveling the temporal and spatial interactions of multiple signaling pathways requires coordinated observation of multiple networks within individual cells and multiple neurons within intact circuits. Large gaps in knowledge remain because our current tools for tracking the dynamics of molecular activity are poorly suited for investigating more than one reporter at a time. Here, we propose to tackle this constraint through development of a novel methodology for simultaneous optical imaging of multiple quantitative FRET biosensors within single neurons, using FLuorescence Anisotropy Reporters (FLAREs). Numerous FLAREs targeting canonical signaling pathways, including calcium, cAMP, and the MAPK cascade, have been constructed in several colors allowing simultaneous imaging of up to three sensors in a single preparation, either in the same or complimentary pathways. We propose three aims to validate and further develop this technology to tailor it for studying cells and circuitry in acute and cultured slices from the mouse brain during neural coding. We will first adapt an optical sectioning microscopy method that is highly advantageous for fluorescence polarization imaging, known as dual-inverted Selective Plane Illumination Microscopy (diSPIM), for FLARE imaging. We will also expand the FLARE palette to include key regulators of synaptic function (Rac, CaMKII) and membrane excitability (voltage). Construction of the FLARE-SPIM instrument will enable proof of principle studies on two high-value neuronal circuits. First, pushing the limits of subcellular spatial resolution, FLARE-SPIM imaging will be performed on key signaling molecules in single dendritic spines in acute hippocampal brain slices during induction of long-term potentiation. Second, pushing the limits of cellular temporal resolution, we will track the rhythmic fluctuations of voltage, calcium, PKA and ERK activities during circadian oscillations of neuronal activity exhibited in organotypically-cultured suprachiasmatic nucleus brain slices. Together, these studies will lay the foundation for systematic exploration of neuromodulation within cells and neuronal circuitry, providing critical and unprecedented new insights for the spatial and temporal interactions between signaling pathways. Through collaboration with other Brain Initiative groups working on similar problems, this foundational work will be scalable to add suites of sensors that visualize nodes of coordinated cellular activity and reveal and measure the complexity of neural coding within intact brain circuits.